The present invention relates to a method for purifying and isolating carboxyl-terminal peptides from its original polypeptide by a simple procedure.
Since the development of the Edman method for sequentially determining the sequence of amino acid residues of a polypeptide from its N-terminal in 1950 (P. Edman, Acta Chem. Scand., 4, 283 (1950)), various improvements have been made in the above method. Today N-terminal sequence analyses can be accomplished even with trace amounts of a sample at a picomolar level using an automatic sequencer (Experimental Techniques in Biochemistry, Second Series, 2. Chemistry of Protein (A), pp. 247-373, Ed. Jpn. Soc. Biochem., 1987, Tokyo Kagaku Dojin).
Meanwhile, with the advent of genetic engineering techniques in recent years, it has become most usual to estimate the whole amino acid sequence of polypeptides by determining the base sequence of a DNA complementary to a messenger RNA. In ordinary circumstances, however, the base sequence of the complementary DNA may be screened by using as a probe an oligonucleotide prepared on the basis of a determined amino acid sequence for a part of a desired polypeptide. In such cases, the screening of the above base sequence could be carried out with greater ease if the probe corresponded to the amino acid sequence close to a carboxy-terminal of the polypeptide. Furthermore, polypeptides may be actually biosynthesized with processing and often lack a part of a whole amino acid sequence coded by the messenger RNA. It is of prime importance, accordingly, to determine both the amino- and carboxyl-terminals of the polypeptide.
As analyses of a carboxyl-terminal amino acid, a hydrazinolysis method, a tritium labeling method and a carboxypeptidase method are currently employed (Experimental Techniques in Biochemistry, Second Series, 2. Chemistry of Protein (A), p. 230, Ed. Jpn. Soc. Biochem., 1987, Tokyo Kagaku Dojin).
Of these methods, the hydrazinolysis method and the tritium labeling method can only determine the amino acid residue at a carboxyl-terminal. Eventually, they can give no useful information for preparing the probe, and afford only data which are unreliable for identifying the position of amino acid residues coded by the messenger RNA. The carboxypeptidase method comprises sequentially cleaving peptide bonds of a polypeptide by various carboxy-peptidases from its carboxyl-terminal and following the time course of the changes in the cleavage thus prepared. This method, consequently, has disadvantages of (1) being cumbersome, (2) requiring relatively a large amount of sample, (3) involving uncertainty in the residue sequencing, and (4) being inadequate for analysis of relatively long-chain polypeptides.